Paraoxonases — poster-children for gene-environment interactions
By John House
Veteran NIEHS grantee Clement Furlong, Ph.D., a leading researcher in the study of insecticide metabolism and professor of genome sciences and medicine at the University of Washington’s School of Medicine, visited NIEHS Sept. 4. Furlong’s presentation, “The Many Facets of Gene-Environmental Interactions of the Paraoxonases,” was hosted by Superfund Research Program Administrator Danielle Carlin, Ph.D.
Furlong shared the work his group and others have been conducting on the paraoxonase (PON) family of genes that are involved in the detoxification of pesticide metabolites and other toxicants, with a focus on how variation in PON genes influences disease susceptibility. Furlong has experimented with ways to manipulate levels of PON proteins as a treatment and prevention strategy and translated his findings into screening tests to improve worker and public safety, as well as potentially advance personalized medicine.
PON1 and organophosphate compounds
The first physiological function of PON1 Furlong examined was its detoxification of organophosphate (OP) compounds. Cytochrome P450 enzymes replace the P=S bond with the more toxic P=O (oxon) bond that binds acetylcholine esterase (AChE) leading to increased ACh and subsequent nervous system toxicity. PON1 exerts neuroprotective effects by hydrolyzing oxons.
As Furlong noted, “Early in the NIEHS Environmental Genome Project, PON1 was cited as an excellent example of gene-environment interaction,” and his subsequent research has born this out. PON1 has many identified single nucleotide polymorphisms (SNPs) that have been characterized. For example, the SNP at -108C results in twice the levels of plasma PON1 over -108T. In addition, the Q192R SNP results in greater catalytic efficiency of PON1. This effect is important given that PON1-Q192, which is less protective, exists in homozygous state for up to 50 percent of some populations.
Additional experiments in his lab, he explained, provided very solid evidence that high levels of paraoxonase were protective against OP exposure, specifically oxon exposure. Major breakthroughs in understanding the function of PON1 came from the PON1 humanized mice generated at the University of California, Los Angeles by Diana Shih, Ph.D., Jake Lusis, Ph.D., and Aaron Tward, Ph.D. By creating PON1 knockout mice and then inserting a transgene coding for the human forms of PON1 with the Q192 and R192 alleles, they demonstrated conclusively the variability in organophosphate toxicity, oxon metabolism and AChE activity between Q192 and R192 alleles.
Additional humanized mouse experiments by Furlong’s group have partially explained the heightened sensitivity observed in young animals and humans to OP insecticides such as chlorpyrifos and diazinon. The levels of plasma PON1 are low at birth and increase over the developmental time period in mice, postnatal day 21, and humans, postnatal year two. When human PON1 transgenes are expressed in mice, they undergo the same developmental time course of expression as endogenous mouse PON1.
PONs and human health
One of the holy grails of genomic research is individually tailored or personalized medicine, which requires accurate and economically feasible functional assays. Furlong’s group has developed a two-substrate arylesterase assay that is able to differentiate between high and low PON1 activity and subsequent OP toxicity susceptibility. This development has assisted the State of Washington’s BChE (Butyrylcholinesterase or plasma cholinesterase) agricultural worker monitoring program in determining OP poisoning susceptibility.
Recent work by a postdoctoral fellow in Furlong’s group, Judit Marsillach, Ph.D., and Ed Hsieh, Ph.D., a postdoctoral fellow with the research team led by Mike MacCoss, Ph.D., has allowed determination of OP exposure with a single blood draw by measuring the percent modification of the active site serine of BChE by OP adducts, thus obviating the requirement for a pre-spray season blood draw. In addition, Furlong’s group has also made progress modifying the PON1 protein for greater efficiency to treat OP poisoning. Furlong closed his talk on the gene-environment interactions of paraoxonases by stressing that epidemiological studies related to the role of genetic variability in PON1 to risk of exposure or disease must include measures of activity to be useful.
(John House, Ph.D., is a postdoctoral fellow in the NIEHS Genetics, Environment, and Respiratory Disease Group.)